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Comparison of the Allosteric Properties of the Co(II)- and Zn(II)-Substituted Insulin Hexamers

Bloom, Curtis R. and Wu, Nancy and Dunn, Alex and Kaarsholm, Niels C. and Dunn, Michael F. (1998) Comparison of the Allosteric Properties of the Co(II)- and Zn(II)-Substituted Insulin Hexamers. Biochemistry, 37 (31). pp. 10937-10944. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20180213-082014495

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Abstract

The positive and negative cooperativity and apparent half-site reactivity of the Co(II)-substituted insulin hexamer are well-described by a three-state allosteric model involving ligand-mediated interconversions between the three states:  T_3T_3‘ ⇌ T_3°R_3° ⇌ R_3R_3‘ [Bloom, C. R., Heymann, R., Kaarsholm, N. C., and Dunn, M. F. (1997) Biochemistry 36, 12746−12758]. Because of the low affinity of the T state for ligands, this model is defined by four parameters:  L_o^A and L_o^B, the allosteric constants for the T_3T_3‘ to T_3°R_3° and the T_3°R_3° to R_3R_3‘ transitions, respectively, and the two dissociation constants for ligand binding to T_3°R_3° and to R_3R_3‘. The d−d electronic transitions of the Co(II)-substituted hexamer give optical signatures of the T to R transition which can be quantified, but the “spectroscopically silent” character of Zn(II) has made previous attempts to describe the Zn(II) species difficult. This work shows that the T to R state conformational transitions of the Zn(II) hexamer can be easily quantified using the chromophore 4-hydroxy-3-nitrobenzoate (4H3N). When the chromophore is bound to the HisB10 sites of the R state, the absorption spectrum of 4H3N is red-shifted, exhibiting strong absorbance and CD signals, whereas 4H3N does not bind to the T state. Hence, 4H3N can be employed as a sensitive indicator of conformation under conditions that do not significantly disturb the T to R state equilibrium. Using 4H3N as an indicator, these studies show that both L_o^A and L_o^B are made less favorable by the substitution of Co(II) for Zn(II); L_o^A is increased by 10-fold while L_o^B by 35-fold, whereas the ligand affinities of the phenolic pockets are unchanged.


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http://dx.doi.org/10.1021/bi980071zDOIArticle
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Additional Information:© 1998 American Chemical Society. Received 9 January 1998. Published online 10 July 1998. This work was supported by a gift from the Novo Research Institute. We thank Ms. Susan E. Danielsen for assistance with the CD work, and we thank Sven E. Harnung for discussions concerning the interpretion of CD spectra.
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Novo Research InstituteUNSPECIFIED
Issue or Number:31
Record Number:CaltechAUTHORS:20180213-082014495
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180213-082014495
Official Citation:Comparison of the Allosteric Properties of the Co(II)- and Zn(II)-Substituted Insulin Hexamers Curtis R. Bloom, Nancy Wu, Alex Dunn, Niels C. Kaarsholm, and Michael F. Dunn Biochemistry 1998 37 (31), 10937-10944 DOI: 10.1021/bi980071z
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:84802
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:13 Feb 2018 20:56
Last Modified:03 Oct 2019 19:22

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